Mechanism and Catalytic Site Atlas

Introduction

The bo3 ubiquinol oxidase has a tightly bound ubiquinol at the QH (high affinity) site, low-spin protoheme b (heme b) and a redox active binuclear centre comprising a copper ion (CuB) and an O-type heme (heme o3). Tyr 288 is near CuB and heme o3, and has a low pKa due to a covalent bond with His 284. In the resting (oxidised or O) state, the heme o3 contains Fe(III) and CuB is Cu(II). A hydroxide ion is bound by Fe and CuB.

A fully reduced ubiquinol docks in a low affinity QL site. From here two electrons are transferred to the tightly bound ubiquinone at the QH site. An electron is transferred from the QH site (via Met 79, Phe 103, heme b, His 421, Phe 420 and His 419) to the binuclear centre, giving a semi-reduced E state. This occurs at the same time as a proton is accepted by the hydroxide to give a water, which leaves. The semiubiquinone at QH is a neutral radical, stabilised by hydrogen bonding between semiubiquinone atom O1 (which is protonated) and Asp 75 and Arg 71. The second electron is transferred from the semiquinone at QH to the binuclear centre (via the same route as the first electron) to give the fully reduced R state, in which the binuclear centre contains Fe(II) and Cu(I). Molecular oxygen then binds to Fe(II), giving a short-lived A state. The next intermediate depends on whether there is an electron available on heme b (which will be delivered by a second ubiquinol docking at site QL, via the same route as before):
(a) If there is an electron available on heme b, four electrons (one from heme b, one from CuB, two from heme o3) reduce oxygen to yield a oxo-ferryl adduct with Fe(IV)=O and Cu(II) (the PR state). Water is produced, with the protons from a pool of water above the active site and/or Tyr 288.

(b) If there is no electron available on heme b, four electrons (one from CuB, two from heme o3, one from Tyr 288) reduce oxygen to yield a oxo-ferryl adduct with Fe(IV)=O, Cu(II) and a Tyr 288 radical (the PM state). Water is produced, with the protons from a pool of water above the active site and Tyr 288.

Both the P states decay to the F state (by protonation of Tyr 288 for PR, and reduction and protonation for PM) which also has Fe(IV)=O and Cu(II) but a regenerated Tyr 288. The second electron from the second QL ubiquinol is delivered to give the resting O state with Fe(III), Cu(II), hydroxide and protonated Tyr 288. The protons for water production and the associated proton pumping are sourced from the cytosolic side of the membrane.

There are two channels, homologous to the K and D channels of the better studied cytochrome c oxidase:

• K channel - from Ser 315, via Ser 299, Lys 362, Thr 359 and the heme o3 hydroxyethylfarnesyl tail, to Tyr 288
• D channel - from Asp 135, via Asn 124, Thr 211, Asn 142, Asn 124, Tyr 61, Thr 204, Ser 145, Thr 201 and Thr 149, to Glu 286.

The K channel is used for two protons required to move from the O state to the R state; the D channel is used for the six other protons required for movement from the A state through to the O state. The mechanism for coupling oxygen reduction and proton pumping is not known and has long been a topic of debate.